Acellular Hb in the circulation has been proposed to be a major source of toxicity and poor outcome for transfusions involving either aged RBCs or HBOCs. One of the major overall objectives of this program project is to test the hypothesis that acellular Hbs contribute to these poor outcomes due to depletion of endothelium-generated NO and that acellular Hb toxicity can be reversed through compensatory mechanisms that generate bioactive NO. Project 3 in conjunction with the other projects will: i) determine the role of bioactive NO in the creation or exacerbation of acellular Hb induced toxicities; and ii) establish of the role of released or nitrite-generated bioactive NO in preventing, limiting, or ameliorating acellular Hb toxicities. Project 3 will contribute to these objectives through the following three Specific Aims: i) Test the hypothesis that a combination of an appropriately modified Hb and added cofactors can produce a formulation that is highly effective in generating long lived forms of bioactive NO; ii) Test the hypothesis that a new biocompatible NO-releasing nanoparticle platform with proven in vivo efficacy as a vasodilator can be further modified to provide for sustained production of both NO and nitrosothiols at therapeutically effective levels suitable for transfusion medicine; and iii) Test the hypothesis that effective generation of bioactive NO from either Hb or nanoparticles can be used to limit or reverse specific oxidative reactions of Hb such as ferryl and tyrosine radical formation that have been implicated in mechanisms of acellular Hb toxicity (Project 2, Core D).